mRNA transcript sythesis from DNA templet.ppt

Describes the sythesis of mRNA transcript from a DNA templet.
Gives detailed regulations of the process

1. Molecular Biology
Fourth Edition
Chapter 15
Messenger RNA
Processing II: Capping
and Polyadenylation
Lecture PowerPoint to accompany
Robert F. Weaver
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2. 15-2
15.1 Capping
•By 1974, mRNA from a variety of
eukaryotic species and viruses were found
to be methylated
•A significant amount of this methylation
was clustered at the 5’-end of mRNA
•This methylation cluster formed a structure
we call a cap

3. 15-3
Cap Structure
•Early study used viral mRNA as they are
easier to purify and investigate
•The -phosphate of a nucleoside
triphosphate remains only in the first
nucleotide in an RNA
–Cap is at the 5’-terminus of RNA
–The cap is made of 7-methylguanosine, m
7
G
–Linkage is a triphosphate
–Charge on the cap area is near -5

4. 15-4
Reovirus Cap Structure
•The m
7
G contributes
a positive charge
•Triphosphate linkage
contributes 3 negative
charges
•Phosphodiester bond
contributes 1 negative
charge
•Terminal phosphate
contributes 2 negative
charges

5. 15-5
Cap Synthesis
•First step
–RNA triphosphatase
removes terminal
phosphate from pre-mRNA
–Then, guanylyl transferase
adds capping GMP from
GTP
•Next, 2 methyl
transferases methylate N
7

of capping guanosine and
2’-O-methyl group of
penultimate nucleotide
•This occurs early in
transcription, before chain
is 30 nt long

6. 15-6
Functions of Caps
Caps serve at least four functions:
–Protect mRNAs from degradation
–Enhance translatability of mRNAs
–Transport of mRNAs out of nucleus
–Efficiency of splicing mRNAs

7. 15-7
15.2 Polyadenylation
•The process of adding poly(A) to RNA is
called polyadenylation
•A long chain of AMP residues is called
poly (A)
•Heterogeneous nuclear mRNA is a
precursor to mRNA

8. 15-8
Poly(A)
•Most eukaryotic mRNAs and their
precursors have a chain of AMP residues
about 250 nt long at their 3’-ends
•Poly(A) is added posttranscriptionally by
poly(A) polymerase

9. 15-9
Functions of Poly(A)
•Poly(A) enhances both the lifetime and
translatability of mRNA
•Relative importance of these two effects
seems to vary from one system to another
•In rabbit reticulocyte extracts, poly(A)
seems to enhance translatability by
helping to recruit mRNA to polysomes

10. 15-10
Basic Mechanism of
Polyadenylation
•Transcription of
eukaryotic genes
extends beyond the
polyadenylation site
•The transcript is:
–Cleaved
–Polyadenylated at 3’-
end created by
cleavage

11. 15-11
Synthesis and Polyadenylation

12. 15-12
Polyadenylation Signals
•An efficient mammalian polyadenylation signal
consists of:
–AAUAAA motif about 20 nt upstream of a
polyadenylation site in a pre-mRNA
–Followed 23 or 24 bp later by GU-rich motif
–Followed immediately by a U-rich motif
•Variations on this theme occur in nature
–Results in variation in efficiency of polyadenylation
–Plant polyadenylation signals usually contain
AAUAAA motif
–More variation exists in plant than in animal motif
–Yeast polyadenylation signals are even more different

13. 15-13
Cleavage of Pre-mRNA
•Polyadenylation involves both:
–Pre-mRNA cleavage
–Polyadenylation at the cleavage site
•Cleavage in mammals requires several proteins
–CPSF – cleavage and polyadenylation specificity
factor
–CstF – cleavage stimulation factor
–CF I
–CF II
–Poly (A) polymerase
–RNA polymerase II

14. 15-14
Initiation of Polyadenylation
•Short RNAs mimic a newly created mRNA 3’-
end can be polyadenylated
•Optimal signal for initiation of such
polyadenylation of a cleaved substrate is
AAUAAA followed by at least 8 nt
•When poly(A) reaches about 10 nt in length,
further polyadenylation becomes independent of
AAUAAA signal and depends on the poly(A)
itself
•2 proteins participate in the initiation process
–Poly(A)polymerase
–CPSF binds to the AAUAAA motif

15. 15-15
Elongation of Poly(A)
•Elongation of poly(A) in mammals requires
a specificity factor called poly(A)-binding
protein II (PAB II)
•This protein
–Binds to a preinitiated oligo(A)
–Aids poly(A) polymerase in elongating poly(A)
to 250 nt or more
•PAB II acts independently of AAUAAA
motif
–Depends only on poly(A)
–Activity enhanced by CPSF

16. 15-16
Polyadenylation Model
•Factors assemble on
the pre-mRNA guided
by motifs
•Cleavage occurs
•Polymerase initiates
poly(A) synthesis
•PAB II allows rapid
extension of the
oligo(A) to full-length

17. 15-17
Poly(A) Polymerase
•Cloning and sequencing cDNAs encoding
calf thymus poly(A) polymerase reveal a
mixture of 5 cDNAs derived from alternative
splicing and alternative polyadenylation
•Structures of the enzymes predicted from
the longest sequence includes:
–RNA-binding domain
–Polymerase module
–2 nuclear localization signals
–Ser/Thr-rich region – this is dispensable for
activity in vitro

18. 15-18
Turnover of Poly(A)
•Poly(A) turns over in the cytoplasm
•RNases tear it down
•Poly(A) polymerase builds it back up
•When poly(A) is gone mRNA is slated
for destruction

19. 15-19
Cytoplasmic Polyadenylation
•Cytoplasmic polyadenylation is most easily
studied using Xenopus oocyte maturation
•Maturation-specific polyadenylation of
Xenopus maternal mRNAs in the
cytoplasm depends on 2 sequence motifs:
–AAUAAA motif near the end of mRNA
–Upstream motif called the cytoplasmic
polyadenylation element (CPE)
•UUUUUAU
•Or closely related sequence

20. 15-20
15.3 Coordination of mRNA
Processing Events
•After reviewing capping, polyadenylation
and splicing, it is clear that these
processes are related
•Cap can be essential for splicing, but only
for splicing the first intron
•Poly(A) can also be essential, but only for
splicing out the last intron

21. 15-21
Effect of Cap on Splicing
•Removal of the first intron fro model pre-mRNAs
in vitro is dependent on the cap
•This effect may be mediated by a cap-binding
complex involved in spliceosome formation

22. 15-22
Effect of Poly(A) on Splicing
•Polyadenylation of model substrates in
vitro is required for active removal of the
intron closest to the poly(A)
•Splicing any other introns out of these
substrates occurs at a normal rate even
without polyadenylation

23. 15-23
mRNA-Processing Occurs
During Transcription
All three of the mRNA-processing events
take place during transcription
–Splicing begins when transcription is still
underway
–Capping
•When nascent mRNA is about 30 nt long
•When 5’-end of RNA first emerges from
polymerase
–Polyadenylation occurs when the still-growing
mRNA is cut at the polyadenylation site

24. 15-24
Binding of CTD of Rpb1 to
mRNA-Processing Proteins
•The CTD of Rpb1 subunit of RNA
polymerase II is involved in all three types
of processing
•Capping, polyadenylating, and splicing
enzymes bind directly to the CTD which
serves as a platform for all three activities

25. 15-25
CTD Phosphorylation
•Phosphorylation state of the CTD of Rpb1 in
transcription complexes in yeast changes as
transcription progresses
–Transcription complexes close to the promoter
contain phosphorylated Ser-5
–Complexes farther from the promoter contain
phosphorylated Ser-2
•Spectrum of proteins associated with the CTD
also changes
–Capping guanylyl transferase is present early when
the complex is close to promoter, not later
–Polyadenylation factor Hrp1 is present in transcription
complexes near and remote from promoter

26. 15-26
RNA Processing Organized by CTD

27. 15-27
Coupling Transcription
Termination with End Processing
•An intact polyadenylation site and active
factors that cleave at the polyadenylation
site are required for transcription
termination
•Active factors that polyadenylate a cleaved
pre-mRNA

28. 15-28
Mechanism of Termination
•Termination of transcription by RNA
polymerase II occurs in 2 steps:
–Transcript experiences a cotranscriptional
cleavage (CoTC) within termination region
downstream of the polyadenylation site
•This occurs before cleavage and polyadenylation
at the poly(A) site
•It is independent of that process
–Cleavage and polyadenylation occur at the
poly(A) site
•Signals polymerase to dissociate from template

29. 15-29
Termination Signal
•CoTC element downstream of the
polyadenylation site in the human b-globin
mRNA is a ribozyme that cleaves itself
–This generates a free RNA 5’-end
–This cleavage is required for normal
transcription termination
–It provides an entry site for Xrn2, a 5’3’
exonuclease that loads onto the RNA and
chases RNA polymerase by degrading the
RNA

30. 15-30
Xrn2, Exonuclease
•Xrn2 terminates transcription like a
“torpedo”
•There is a similar torpedo mechanism in
yeast where cleavage at poly(A) site
provides entry for the 5’3’ exonuclease
Rat1
•Rat1 degrades the RNA until it catches the
polymerase and terminates transcription

31. 15-31
Torpedo Model for Transcription
Termination

32. 15-32
Role of Polyadenylation in
mRNA Transport
•Polyadenylation is required for efficient
transport of mRNAs from their point of
origin in the nucleus to the cytoplasm